In the research field of molecular biology, and clinical application field such as genetic testing, a method for specifically amplifying the target nucleic acid piece has been a very important technology. Examples of such a gene amplification method include developing isothermal amplification methods, such as LAMP (Loop-Mediated Isothermal Amplification) method and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, in addition to the most general PCR method (Polymerase Chain Reaction).
The method for detecting the specific nucleic acid region amplified by a method such as PCR method, LAMP method, or ICAN method can be classified into two methods: a method for detecting DNA, an amplification product; and a method for detecting a pyrophosphoric acid (diphosphoric acid), a by-product.
As the most general method for detecting a double-stranded nucleic acid, known is a method for subjecting a solution after amplification reaction to Agarose electrophoresis, and applying the solution with fluorescent intercalators such as Ethidium Bromide or SYBR Green, and observing specific fluorescence (Non-Patent Document 1). However, a method for dyeing with fluorescent intercalators such as Ethidium Bromide after electrophoresis takes about 30 minutes to 1 hour for electrophoresis and needs expensive machines such as a UV irradiator or a fluorescent detector for detection of fluorescence.
As a method for detecting and determining a PCR product without performing electrophoresis, known is a method wherein fluorescent intercalators are added primarily to a reaction liquid before starting PCR, fluorescence intensity is measured with a fluorospectrophotometer, and the amount of amplified DNA is determined (Patent Document 1). However, since the fluorescent intercalators bind to single-stranded nucleic acid such as primer, a background signal which is independent of the amount of double-stranded nucleic acid is enhanced, resulting in reduction in detection sensitivity. In contrast, there is also known a method for reducing the intensity of background signals by treating compounds that preferentially react with intercalators bound to single-stranded nucleic acid (Patent Document 2). However, use of these methods involves devices and facilities for detecting fluorescence.
As a method other than the above methods, for example, known is a method for conducting nucleic acid amplification reaction with fluorescence-labeled primer, and detecting the nucleic acid amplification product with fluorescence polarization (Patent Document 3). However, the method needs a complicated operation for separating the fluorescence-labeled primer which was not incorporated into an amplification product, and the separation operation of the primer decrease the yield of the obtained nucleic acid amplification fragments, which may consequently cause reduction in detection sensitivity. Also, labeled nucleotide and labeled primer are usually very expensive in terms of cost.
Also known is a method for detecting a nucleic acid amplification product by passing polarized light through reaction liquid in nucleic acid amplification reaction and measuring the angle of rotation or circular dichroism of the polarized light (Patent Document 4). However, the measurement of angle of rotation and circular dichroism needs a special device.    Patent Document 1: Japanese Kokai Publication H5-237000    Patent Document 2: WO 2002/103053    Patent Document 3: Japanese Kokai Publication H9-187275    Patent Document 4: Japanese Kokai Publication 2002-186481    Non-Patent Document 1: Molecular Cloning second edition, vol. 1, 6.15 (1989)